Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein alpha-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not.
Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.
Dean
D R
DR
Department of Anaerobic Microbiology, Virginia Polytechnic Institute, State University, Blacksburg 24061.
Setterquist
R A
RA
Brigle
K E
KE
Scott
D J
DJ
Laird
N F
NF
Newton
W E
WE
D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D008983:Molybdoferredoxin; D006639:Histidine; D009591:Nitrogenase; D003545:Cysteine